Chromobacterium species with insecticidal activity

ABSTRACT

Strains of  Chromobacterium sphagni  sp. nov. are described which have insecticidal activity against insect larvae, in general, and lepidopteran insect larvae, in particular. A biocontrol agent containing one or more  C. sphagni,  media in which the  C. sphagni,  or both, and optionally a carrier are also described. Methods of killing insect larvae and methods of reducing insect populations in an area by applying to the area or an object an effective amount of the biocontrol agent are also described.

CROSS REFERENCE TO RELATED APPLICATION

This application is related to and claims priority to U.S. PatentApplication 62/304,594 filed on Mar. 7, 2016, the contents of which areincorporated herein.

BACKGROUND OF THE INVENTION

Sequence Listing

The Sequence Listing submitted via EFS-Web as ASCII compliant text fileformat (.txt) filed on Mar. 3, 2017, named “SequenceListing_ST25”,(created on Feb. 27, 2017, 7 KB), is incorporated herein by reference.This Sequence Listing serves as paper copy of the Sequence Listingrequired by 37 C.F.R. §1.821(c) and the Sequence Listing incomputer-readable form (CRF) required by 37 C.F.R. §1.821(e). Astatement under 37 C.F.R. §1.821(f) is not necessary.

Field of the Invention

This invention relates to a novel species of Chromobacterium, which isbeing named Chromobacterium sphagni sp. nov., that has insecticidalactivity. This invention also relates to compositions containing thesenovel Chromobacterium strains and use of these compositions to killinsect larvae.

Description of Related Art

Lepidoptera is an order of insects that includes moths and butterflies.The larvae of many Lepidoptera are economic pests for agriculturebecause they feed on many different types of crops. While many birds andother animals feed on Lepidoptera, these predators do not sufficientlyreduce the harm caused by the larvae to food crops. Parasitic wasps andflies also help reduce the lepidopteran populations, but again notsufficiently to reduce economic damage to crops. On the other hand,insecticides can be used to destroy populations of Lepidoptera. But manyof the insecticides are non-specific and harm beneficial animals,especially bees and birds. Further, the pesticides can enter groundwaterand be ingested by humans, again causing harm. As such, a need existsfor compositions that can kill lepidopteran larvae and methods of usingthe compositions to kill lepidopteran larvae, thereby protecting crops.

Other insects also destroy economically valuable plants (crops, trees,ornamental plants) by feeding on the economically valuable plants eitherwhile in larvae stage or as an adult. It is difficult to kill many theseinsects or reduce their population without using pesticides that areharmful to the plant or mammals that eat the plants.

Until recently, purple-pigmented bacteria of the genus Chromobacteriumwere represented by a single species, Chromobacterium violaceum(Bergonzini 1881). Chromobacterium violaceum is best known forproduction of the purple pigment violacein, which has exhibited diverseantimicrobial and antitumor activities. In 2007, Martin, et al., Int. J.Systemic and Evolutionary Micro 57:993-999 (2007) describedChromobacterium subtsugae, which in addition to violacein, also producedinsecticidal factors that were active against a variety of insect pests.See also U.S. Pat. No. 7,244,607. More recently, an extract of C.subtsugae was approved by the EPA for use as an organic insecticide thatis now commercially available as Grandevo® (Marrone BioInnovations,Davis, Calif.). Since the description of C. subtsugae, five additionalspecies of Chromobacterium have been described: C. aquaticum, C.haemolyticum, C. piscinae, C. pseudoviolaceum and C. vaccinii.Chromobacterium vaccinii also produces insecticidal factors that killmosquito larvae and other insect larvae. See WO 2015/020848.

Bacillus thuringiensis var. kurstaki (Btk) is an effective biocontrolagent for lepidoteran insects that has been in use since its discoveryin 1962. However some species of Lepidoptera have recently becomeresistant to the Cry toxin produced by Btk. See, Cancino-Rodenzo, etal., Insect Biochem. Mol. Biol. 40:58-63 (2010). Thus, a need exists foranother biocontrol agent that can selectively kill insects in general,and more specifically lepidopteran species.

BRIEF SUMMARY OF THE INVENTION

It is an object of this invention to have a biocontrol agent useful forkilling insect larvae. This biocontrol agent contains an insecticidalcomposition which can be any Chromobacterium sphagni, media in which C.sphagni grew, or a combination thereof. It is a further object of thisinvention that the C. sphagni can be C. sphagni strain 14B-4 (NRRLB-67382), C. sphagni strain 37-2 (NRRL B-67131), C. sphagni strain14B-5, C. sphagni strain 14B-6, C. sphagni strain 36-1, C. sphagnistrain 36-2, C. sphagni strain 36-3, C. sphagni strain 36-4, C. sphagnistrain 36-5, C. sphagni strain 36-6, C. sphagni strain 37-1, C. sphagnistrain 37-3, C. sphagni strain 37-4, C. sphagni strain 37-5, C. sphagnistrain 37-6, and a combination thereof. It is a further object of thisinvention that the biocontrol agent contains a carrier. This biocontrolagent can be a pellet, wettable powder, dust, granule, adherent dust orgranule, solution, emulsifiable concentrate, emulsion, suspensionconcentrate, aerosol, and/or bait. It is also an object of thisinvention that the carrier can be food which insect larvae eat, water,one or more surfactants, one or more emulsifiers, one or more alcohols,one or more oils, one or more glycerols, one or more biological buffers,one or more ethers, one or more glycols, one or more ketones, one ormore esters, one or more clays, one or more silicas, one or morecellulosics, one or more rubber, one or more synthetic polymers, or acombination thereof. The biocontrol agent can be further formulated withinsect attractants, such as pheromones, an insect extract containingpheromones, or other non-pheromone compounds known to attract the targetinsects; adjuvants, adhesives, and/or dispersants.

It is an object of this invention to have a method for killing insectlarvae by applying the biocontrol agent of this invention in an amounteffective to kill the insect larvae to an area in which the insectlarvae are present or onto an object in said area. Another object ofthis invention is that the biocontrol agent kills the insect larvaeafter the insect larvae ingest the biocontrol agent. In one embodimentof this invention, the object is a plant on which insect larvae live orwhich the insect larvae eat. In a further object of this invention, theinsect larvae are lepidopteran insect larvae. It is also an object ofthis invention that the carrier in the biocontrol agent can be foodwhich insect larvae eat, water, one or more surfactants, one or moreemulsifiers, one or more alcohols, one or more oils, one or moreglycerols, one or more biological buffers, one or more ethers, one ormore glycols, one or more ketones, one or more esters, one or moreclays, one or more silicas, one or more cellulosics, one or more rubber,one or more synthetic polymers, or a combination thereof. Another objectof the invention is that the biocontrol agent can also contain one ormore insect larvae attractants, one or more adjuvants, one or morepheromones, one or more adhesives, one or more dispersants, or acombination thereof.

It is an object of this invention to have a method of reducing thepopulation of insect larvae by applying the biocontrol agent of thisinvention to an area in which the insect larvae are present or onto anobject in the area in an amount effective to kill the insect larvae(after the larvae ingest the biocontrol agent) thereby reducing thepopulation of said insect larvae. It is an optional object of thisinvention that the object is a plant on which the insect larvae live oreat. It is another object of this invention that the insect larvae arelepidopteran insect larvae. It is also an object of this invention thatthe carrier in the biocontrol agent can be food which insect larvae eat,water, one or more surfactants, one or more emulsifiers, one or morealcohols, one or more oils, one or more glycerols, one or morebiological buffers, one or more ethers, one or more glycols, one or moreketones, one or more esters, one or more clays, one or more silicas, oneor more cellulosics, one or more rubber, one or more synthetic polymers,or a combination thereof. Another object of the invention is that thebiocontrol agent can also contain one or more insect larvae attractants,one or more adjuvants, one or more pheromones, one or more adhesives,one or more dispersants, or a combination thereof.

It is a further object of this invention to have a compositioncontaining C. sphagni and media in which the C. sphangi grew. It isanother object of this invention that the composition contains betweenapproximately 10³ CFU to approximately 10¹⁰ CFU C. sphagni. It isanother object of this invention that the composition can contain morethan one strain of C. sphagni, including but not limited to, C. sphagnistrain 14B-4 (NRRL B-67382), C. sphagni strain 37-2 (NRRL B-67131), C.sphagni strain 14B-5, C. sphagni strain 14B-6, C. sphagni strain 36-1,C. sphagni strain 36-2, C. sphagni strain 36-3, C. sphagni strain 36-4,C. sphagni strain 36-5, C. sphagni strain 36-6, C. sphagni strain 37-1,C. sphagni strain 37-3, C. sphagni strain 37-4, C. sphagni strain 37-5,and C. sphagni strain 37-6. It is further object of this invention thatthe C. sphagni has an 16S rRNA gene sequence of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO: 3. It is another object of this invention that thecombined 16:1 w7c/16:1 w6c fatty acids in C. sphagni ranges fromapproximately 43% to approximately 47%. It is a further object of thisinvention to use this composition to kill insect larvae that ingest thiscomposition.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the maximum likelihood analysis of 16S rRNA gene sequencesfrom Chromobacterium sphagni sp. nov. 37-2 with those of recognizedChromobacterium species. Vogesella indigofera is included as anoutgroup.

STATEMENT REGARDING DEPOSIT OF BIOLOGICAL MATERIAL UNDER THE TERMS OFTHE BUDAPEST TREATY

The inventors deposited samples of novel Chromobacterium sphagni sp.nov. strain 37-2 as described herein on or before Sep. 10, 2015, andChromobacterium sphagni sp. nov. strain 14B-4 as described herein on orbefore Feb. 15, 2017, with the U.S.D.A., Agricultural Research Service'sPatent Culture Collection located at the National Center forAgricultural Utilization Research, 1815 N. University Street, Peoria,Ill. 61604, in a manner affording permanence of the deposit and readyaccessibility thereto by the public if a patent is granted. The depositshave been made under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure and the regulations thereunder. For strain14B-4, the deposit's accession number is NRRL B-67382 and for strain37-2 the deposit's accession number is NRRL B-67131.

All restrictions on the availability to the public of Chromobacteriumsphagni sp. nov. strains 14B-4 (NRRL B-67382) and 37-2 (NRRL B-67131)which have been deposited as described herein will be irrevocablyremoved upon the granting of a patent covering these particularbiological materials.

The Chromobacterium sphagni sp. nov. strains 14B-4 (NRRL B-67382) and37-2 (NRRL B-67131) have been deposited under conditions such thataccess to the microorganisms are available during the pendency of thepatent application to one determined by the Commissioner to be entitledthereto under 37 C.F.R. §1.14 and 35 U.S.C §122.

The deposited biological materials will be maintained with all the carenecessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of the deposited microorganisms, and in any case, for a period ofat least thirty (30) years after the date of deposit for the enforceablelife of the patent, whichever period is longer.

We, the inventors for the invention described in this patentapplication, hereby declare further that all statements regarding thisDeposit of the Biological Material made on information and belief arebelieved to be true and that all statements made on information andbelief are believed to be true, and further that these statements aremade with knowledge that willful false statements and the like so madeare punishable by fine or imprisonment, or both, under section 1001 ofTitle 18 of the United States Code and that such willful falsestatements may jeopardize the validity of the instant patent applicationor any patent issuing thereon.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are two strains of a novel Chromobacterium species,namely, Chromobacterium sphagni sp. nov. strain 14B-4 (NRRL B-67382) andstrain 37-2 (NRRL B-67131) and cultures in which the bacteria grew.Chromobacterium sphagni sp. nov. possess eight copies of the 16S rRNAgene. The full length 16S rRNA gene sequences of C. sphagni sp. nov.strain 37-2 (NRRL B-67131), determined by sequencing the entire genome,are in SEQ ID NO: 1 and 2 (nucleotide 1452 can be either T or C,respectively). A fragment of the 16S rRNA gene from C. sphagni sp. nov.strain 14B-4 (NRRL B-67382) is in SEQ ID NO: 3 and spans nucleotides 60to 1491 of SEQ ID NOs: 1 and 2. SEQ ID NO: 3 for C. sphagni sp. nov.strain 14B-4 (NRRL B-67382) contains a T at 16S rRNA gene nucleotide 456(located at position 397 in SEQ ID NO: 3) instead of a C at 16S rRNAgene at nucleotide 456 for SEQ ID NOs: 1 and 2 (located at position 456in SEQ ID NOs: 1 and 2). Further, SEQ ID NO: 3 (strain 14B-4) contains aC at 16S rRNA gene nucleotide 1452 (located at position 1393 in SEQ IDNO: 3); whereas it is either a C or T at this location in SEQ ID NOs: 1and 2 (strain 37-2). These bacteria produce one or more compounds thatcan kill insect larvae in general, and, more specifically, lepidopteraninsect larvae. The C. sphagni sp. nov. strains described herein wereobtained from two distinct sphagnum bogs. See Example 1, infra, for moreinformation. Not wishing to be bound to any particular hypothesis, theC. sphagni sp. nov. strains 14B-4 (NRRL B-67382) and 37-2 (NRRL B-67131)synthesize one or more compounds which are toxic to insect larvae.Because these C. sphagni produce one or more compounds that are toxic toinsect larvae, the bacteria and/or the media in which the bacteria greware considered the insecticidal compositions of the biocontrol agentsdescribed herein. C. sphagni sp. nov. strain 14B-4 (NRRL B-67382) and C.sphagni sp. nov. strain 37-2 (NRRL B-67131) are representative strainsof this invention. This invention includes any C. sphagni, isolated froma sphagnum bog and identified by its 16S rRNA gene sequence beingsimilar to SEQ ID NOs: 1, 2 or 3, media containing any such C. sphangi,and the use of any such C. sphangi or media containing the bacteria, asdescribed herein. In addition to strains 14B-4 and 37-2, the C. sphagniof this invention includes strains 14B-5, 14B-6, 36-1, 36-2, 36-3, 36-4,36-5, 36-6, 37-1, 37-3, 37-4, 37-5, 37-6, and a combination thereof.

Described herein are methods of killing insects, involving exposing (ortreating) the insect larvae to the biocontrol agents described herein byapplying the biocontrol agent to an object (e.g., insects, plants, fruittrees, screens and netting, traps) or an area (e.g., water, soil, house,farm land) in need of such treatment. The amount of the biocontrol agentto be applied should be sufficient to kill the insect larvae (aneffective amount). Also described herein are methods of reducing insectpopulations by applying an effective amount of the biocontrol agent toan object or area.

The terms “object” or “area” as used herein include any place where thepresence of target pests is not desirable, including any type of tree,crop, natural or artificial parkland, watercourse, or other target pesthabitat. In one embodiment, the area or object where the biocontrolagent can be dispersed, placed, applied, etc., excludes sphagnum bogs.

The terms “object” or “area” as used herein include any place where thepresence of target insect pests (e.g., Lepidoptera species) are notdesirable, including any type of premises, which can be out-of-doors,such as in gardens, lawns, tents, camping areas, farmland, parks, etc.,or indoors, such as in barns, garages, commercial buildings, homes,etc., or any area where insect pests are a problem, such as in shippingor storage containers (e.g., bags, boxes, crates, etc.), packingmaterials, bedding, and so forth. Also included in the definition ofobject or area are the outer covering of a living being, such as skin,fur, hair, or clothing. Thus, the methods include dispensing thebacteria, media in which the bacteria were grown, or biocontrol agentsdescribed herein into the area in traps, sprays, emulsions, freeze-driedblocks, coatings or vapor form (e.g., an aerosol). One may use devicesthat allow a slow sustained release of bacteria, media in which thebacteria were grown, and/or biocontrol agent into the environment from asealed canister or chemical or physical (e.g., fabric) matrix. One mayalso use a biocontrol agent that contains a bait for the insect pest andcontains the bacteria and/or media in which the bacteria were grown. Thebiocontrol agent can be placed in an area or on an object where insectpests are not wanted and in a manner that the larvae of the insect pestsingest the biocontrol agent. Similarly, the biocontrol agent can containa formulation of the media in which the bacteria were grown and sprayedonto economically important plants (crops, trees, ornamental plants,etc.) or onto other objects in a manner such that the larvae of theinsect pests will ingest the biocontrol agent. In one embodiment, plantsthat live in sphagnum bogs are not considered economically importantplants.

One applies, at a minimum, an effective amount of a biocontrol agentcontaining C. sphangi and/or media containing the bacteria, as describedherein. C. sphagni includes, but is not limited to strains 14B-4 (NRRLB-67382), 14B-5, 14B-6, 36-1, 36-2, 36-3, 36-4, 36-5, 36-6, 37-1, 37-2(NRRL B-67131), 37-3, 37-4, 37-5, 37-6, and a combination thereof. Theterm “effective amount,” as used herein, means the minimum amount of thecompositions needed to kill the insects when compared to the same areaor object which is untreated. The precise amount needed will, bynecessity, vary in accordance with the target insect; particularcomposition used; the type and size of area or object to be treated;weather or climatic conditions under which it is applied; and theenvironment in which the area or object is located. The precise amountof the composition can easily be determined by one skilled in the artgiven the teachings herein. In one embodiment, an effective amountcontains between approximately 10³ to approximately 10¹⁰ CFU of at leastone bacterium of this invention. In another embodiment, effective amountcontains between approximately 10⁵ to approximately 10⁹ CFU of at leastone bacterium of this invention. In another embodiment, effective amountcontains between approximately 10⁷ to approximately 10⁹ CFU of at leastone bacterium of this invention.

The biocontrol agents described herein which contain an insecticidalcomposition and optionally a carrier. The insecticidal composition cancontain one or more C. sphagni strains discussed herein. Theinsecticidal composition may also contain media in which the bacteriawere grown, alone, or with the bacteria that were cultured in thatmedia. The media may contain the bacteria or the cytosolic components ofthe bacteria. In one embodiment, the bacteria are grown in media untilthe bacteria have reached their peak growth and the number of livebacteria is decreasing. Such media is sometimes referred to as “spentmedia”. As such, the insecticidal composition, in one embodiment,contains spent media with the bacteria described herein. In anotherembodiment, the bacteria are lysed and the media is filtered to removebacterial cell wall and membrane components. In such an embodiment, theinsecticidal composition contains the spent media without wholebacteria. In yet another embodiment, the bacteria are inactivated andleft in the media. In such an embodiment, the insecticidal compositioncontains the media and the inactivated bacteria. In another possibleembodiment, the bacteria are not actively lysed, but the bacteria themedia are still separated from each other. Then one can use the media orthe bacteria in the biocontrol agent. In any of these embodiments, themedia can be applied in a liquid-form or freeze-dried and applied as asolid, or freeze-dried and then resuspended in another liquid or reducedvolume. One another embodiment, the biocontrol agent may also containone or more other compounds (e.g., insect larvae attractants, adjuvants,pheromones, adhesives, dispersants or other insecticidal agents known inthe art) provided the one or more other compounds do not substantiallyinterfere with the insecticidal activity or efficacy of the insecticidalcomposition described herein. Whether or not one or more other compoundsinterfere with the insecticidal activity and/or efficacy of theinsecticidal compositions can be determined, for example, by theprocedures utilized below. The biocontrol agent, in one embodiment,excludes liquids and plants obtained from a sphagnum bog. In anotherembodiment, the biocontrol agent of the present invention excludeskilled insects or insects that have already ingested the bacteriadescribed herein. In another embodiment, the biocontrol agent describedherein excludes water, insect bait, or other substances obtained from asphagnum bog. “Non-sphagnum bog water” is water that is not obtainedfrom a sphagnum bog.

The biocontrol agent described herein can contain a carrier. The carriermay be, for example, any agronomically or physiologically orpharmaceutically acceptable carrier. The carrier as used herein isdefined as not including the body of an insect. One carrier is insectfood. The carrier in another embodiment can be an insect attractant suchas bait or pheromones. The carrier should not be harmful to plantsand/or other non-target organisms. In some embodiments, the biocontrolagent can be insect food impregnated with one or more of theinsecticidal composition described herein, or the insecticidalcomposition described herein can be sprayed onto insect food. One canapply such embodiments of the biocontrol agent to areas where the insectlarvae live. In another embodiment, the carrier can be media in whichsaid C. sphagni grows or grew.

The bacteria and/or media are optionally used in combination with one ormore carriers or additives such as water, humectants, surfactants, inertcarriers, other insecticides, and colorants; typical humectants, inertcarriers, insecticides, and colorants are well known in the art. As apractical matter, it is expected that the bacteria will be formulatedwith an inert carrier for use as a pesticide composition. Such inertcarriers are well known in the art. Water is one inert carrier, althoughother inert carriers suitable for use herein include but are not limitedto inorganic or organic biological buffers, alcohols, ethers, glycols,ketones, esters, and solid carriers such as clays, silicas, cellulosics,rubber, or synthetic polymers. Surfactants are well-known in the artfield and used to assist in the mixing of, for example, freeze-driedmedia containing bacteria and water.

A single application will suffice under optimum conditions, withmortality occurring rapidly, but under suboptimum conditions, eitherhigher concentrations or multiple applications may be necessary.

The biocontrol agent described herein contains at least one C. sphagniand optionally one or more carriers. C. sphagni includes, but is notlimited to, strains 14B-4 (NRRL B-67382), 14B-5, 14B-6, 36-1, 36-2,36-3, 36-4, 36-5, 36-6, 37-1, 37-2 (NRRL B-67131), 37-3, 37-4, 37-5,37-6, and a combination thereof.

In one embodiment, the biocontrol agent described herein also containsanother insecticide effective for controlling Lepidoptera species and/orother insect species. As used herein, the term “insecticide” refers to amaterial or mixture of materials which induce mortality, disrupt orimpede growth, interfere with metamorphosis or other morphogenicfunctions, effect sterilization, or interfere with feeding, metabolism,respiration, locomotion or reproduction of the targeted insects.Suitable insecticides include but are not limited to biological controlssuch as insect growth regulators, and materials that are toxic toinsects (i.e., toxicants) such as chemical insecticides, pathogenicnematodes, fungi, protozoans, or other bacteria. In one embodiment,insecticides are slow-acting (i.e., acting over a course of hours, days,weeks, or preferably months) to reduce “avoidance” effects beforeindividuals have distributed the insecticide to other members of thepopulation or colony. Slow-acting insecticides are known in the art. Thecomposition may also contain biological control agents such as toxinsderived from bacteria, fungi, or other organism. One example of abiological control agent is B. thuringiensis toxin.

The biocontrol agents described herein may be formulated as wettablepowders, dusts, granules, adherent dusts or granules, solutions,emulsifiable concentrates, emulsions, suspension concentrates, aerosols,and/or baits. The biocontrol agents may also be further formulated withinsect attractants, such as pheromones, insect extracts containingpheromones, or other non-pheromone compounds known to attract the targetinsects.

The target insects include, but are not limited to, lepidopteraninsects, such as gypsy moth (Lymantria dispar), diamondback moth(Plutella xylostella), tobacco hornworm (Manduca sexta), cabbage looper(Trichoplusia ni), corn earworm (Helicoverpa zea), beet armyworm(Spodoptera exigua), fall armyworm (Spodoptera frugiperda), Europeancorn borer (Ostrinia nubilalis), and tobacco budworm (Heliothisvirescens).

The terms “approximately” and “about” refer to a quantity, level, valueor amount that varies by as much as 30% in one embodiment, or in anotherembodiment by as much as 20%, and in a third embodiment by as much as10% to a reference quantity, level, value or amount. As used herein, thesingular form “a”, “an”, and “the” include plural references unless thecontext clearly dictates otherwise. For example, the term “a bacterium”includes both a single bacterium and a plurality of bacteria of the samespecies.

Many techniques involving molecular biology discussed herein arewell-known to one of ordinary skill in the art and are described in,e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual 4th ed.2012, Cold Spring Harbor Laboratory; Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, 1994-current, John Wiley & Sons; andKriegler, Gene Transfer and Expression: A Laboratory Manual (1993).Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biologymaybe found in e.g., Benjamin Lewin, Genes IX, Oxford University Press,2007 (ISBN 0763740632); Krebs, et al. (eds.), The Encyclopedia ofMolecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9);and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN1-56081-569-8).

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples and theaccompanying drawings, which are included herein only to furtherillustrate the invention and are not intended to limit the scope of theinvention as defined by the claims. The examples and drawings describeat least one, but not all embodiments, of the inventions claimed.Indeed, these inventions may be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure willsatisfy applicable legal requirements.

EXAMPLE 1 Isolation of Bacteria

Two new strains of Chromobacterium sphagni sp. nov. that fulfill thecriteria for a new species, strains 14B-4 (NRRL B-67382) and 37-2 (NRRLB-67131), have been obtained. C. sphagni sp. nov. strain 14B-4 (NRRLB-67382) was isolated from a sphagnum bog in the Allegheny Mountains ofRandolph County, W. Va. C. sphagni sp. nov. strain 37-2 (NRRL B-67131)was isolated from similar habitat (sphagnum bog) in Androscoggin County,Me. The following thirteen additional isolates of Chromobacteriumsphagni sp. nov. with 16S sequences identical to those of SEQ ID NO: 1,SEQ ID NO: 2 and/or SEQ ID NO: 3, were collected from the same sites asC. sphagni sp. nov. strain 14B-4 (NRRL B-67382) and C. sphagni sp. nov.strain 37-2 (NRRL B-67131): 14B-5, 14B-6, 36-1, 36-2, 36-3, 36-4, 36-5,36-6, 37-1, 37-3, 37-4, 37-5 and 37-6. The distribution of C. sphagniappears restricted to sphagnum bogs, and numbers of C. sphagni recoveredfrom sphagnum bogs suggest that they are not abundant bacteria. Onlythrough the use of a selective medium, described below, was it possibleto inhibit the growth of other bacteria sufficiently to isolate C.sphagni, thus indicating that the concentration of C. sphagni strainswithin the sphagnum bogs are very low, compared to the concentration ofother bacteria. Further, these bacteria have not been isolated in othertypes of wetlands. Sphagnum bogs are characterized as wetlands thataccumulate peat, primarily from sphagnum moss and other acid-tolerantplants, and have high concentrations of tannins. The sphagnum bogs inthe Allegheny Mountains of Randolph County, W. Va., and in AndroscogginCounty, Me. are typically cold, in light of their location (highaltitude and high latitude, respectively).

Bacteria are isolated from water samples using a solid medium, modifiedfrom Keeble and Cross (J. App. Microbio. 43:2 325-327 (1977)),containing 1 g yeast extract, 3 g nutrient broth, 10 g glucose, 18 gagar, and 50 mg each of the antibiotics neomycin and cycloheximide perliter of water. Plates are incubated at 24° C. for 48 hours to 72 hours.Potential colonies of Chromobacterium are selected based on violetcolony color. Bacteria used for insect bioassays are then grown in aliquid medium with the same components listed above but without agar orantibiotics. Liquid cultures are shaken at 200 rpm and 24° C. for 96hours.

EXAMPLE 2 Genetic and Biochemical Analyses of Bacteria

Whole genome sequencing of C. sphagni sp. nov. strain 37-2 (NRRLB-67131) on a PacBio RS II long-read sequencer (Pacific Biosciences,Menlo Park, Calif.) reveal that the strain possesses eight copies of the16S rRNA gene, like other members of the genus Chromobacterium, and thatnucleotide 1452 could be either T (SEQ ID NO: 1; 5 gene copies) or C(SEQ ID NO: 2; 3 gene copies). Conventional DNA sequencing of PCRamplified fragments of 16S rRNA genes from C. sphagni sp. nov. strain14B-4 (NRRL B-67382) yield the sequence of SEQ ID NO: 3, which spansnucleotides 60 to 1491 of SEQ ID NO: 1 and SEQ ID NO: 2. SEQ ID NO: 3shows that C. sphagni sp. nov. strain 14B-4 (NRRL B-67382) has asequence identical to nucleotides 60 to 1491 of SEQ ID NOs: 1 and 2,obtained from C. sphagni sp. nov. strain 37-2 (NRRL B-67131), exceptthat nucleotide 456 for 16S rRNA genes in SEQ ID NOs: 1 and 2 are C, andthe corresponding nucleotide (located at the same position within 16SrRNA genes) for strain 14B-4 is T (which is located at position 397within SEQ ID NO: 3 because SEQ ID NO: 3 starts at nucleotide 60 of SEQID NOs: 1 and 2). Further, SEQ ID NO: 3 contains a C at 16S rRNA genenucleotide 1452 (located at nucleotide 1393 in SEQ ID NO: 3); whereas itis either a C or T at this location in SEQ ID NOs: 1 and 2. A comparisonof 16S rRNA gene sequence of C. sphagni sp. nov. strain 37-2 (NRRLB-67131), using SEQ ID NO:1, with the 16S rRNA gene sequences of otherChromobacterium species (C. violaceum, C. subtsugae, C. vaccinii, C.piscinae, C. aquaticum, C. pseudoviolaceum and C. haemolyticum)indicates a lesser degree of relatedness, with C. piscinae displayingthe fewest (19) nucleotide substitutions and C. aquaticum displaying themost (45) substitutions.

Maximum likelihood analysis of the sequences described above (using SEQID NO: 1) can be used to create a depiction of how C. sphagni sp. nov.strain 37-2 (NRRL B-67131) is related to other currently recognizedChromobacterium species. See FIG. 1.

In recent years, the classical DNA-DNA hybridization techniques used todifferentiate bacterial species from each other have been replaced bythe average nucleotide identity (ANI) method described by Richter andRosello-Mora (Proc. Natl. Acad. Sci. USA 106(45):19126-31 (2009)), whichis based on comparisons of genomic sequence data. When comparing twobacteria, an ANI below 95% is considered to indicate distinct species.Comparing C. sphagni sp. nov. strain 37-2 (NRRL B-67131) with availablegenomes of other Chromobacterium species results in ANI values wellbelow 95%, indicating that C. sphagni sp. nov. strain 37-2 (NRRLB-67131) constitutes a separate species. Specifically, comparing thegenome of C. sphagni sp. nov. strain 37-2 (NRRL B-67131) against theinsecticidal species C. subtsugae and C. vaccinii results in ANI valuesof 88.5% and 87.0%, respectively. ANI cannot be calculated for C.sphagni sp. nov. strain 37-2 (NRRL B-67131) vs. C. aquaticum, becausethere is no available genome sequence for C. aquaticum. However, the 16Ssequence for C. aquaticum appears more phylogenetically distant from C.sphagni sp. nov. strain 37-2 (NRRL B-67131) than several species thatare clearly distinct from these isolates based on ANI values.

Table 1, below, shows ANI values for Chromobacterium sphagni sp. nov.strain 37-2 (NRRL B-67131) vs. other Chromobacterium species. Genomicalignments were completed using the MuMmer algorithm.

Like C. vaccinii MWU205^(T) and C. subtsugae PRAA4-1^(T) , C. sphagnisp. nov. strain 37-2 (NRRL B-67131) produces arginine dihydrolase andprotease, assimilates glucose, N-acetylglucosamine and gluconate, doesnot produce urease, B-glucosidase or B-galactosidase, and does notassimilate arabinose, mannitol, maltose, adipic acid or phenylaceticacid. C. sphagni sp. nov. strain 37-2 (NRRL B-67131) differs from C.vaccinii MWU205^(T) in that it is not capable of assimilating malate orcitrate. C. sphagni sp. nov. strain 37-2 (NRRL B-67131) differs from C.subtsugae PRAA4-1^(T) in that it is capable of reducing nitrate tonitrite. Nitrate reductase genes are not detected in the genome sequenceof PRAA4-1, but are found in the genome of C. sphagni sp. nov. strain37-2 (NRRL B-67131).

Fatty acid analysis of C. sphagni sp. nov. strains 14B-4 (NRRL B-67382)and 37-2 (NRRL B-67131) using the MIDI system reveals that the combined16:1 w7c/16:1 w6c fatty acids accounted for 46.9% and 46.2% of totalfatty acid content respectively. The fraction of these particular lipidsare significantly higher than the levels reported for otherChromobacterium species (C. subtsugae 41.9%; C. vaccinii, 41.9%; C.violaceum, 35.8%; C. aquaticum, 33.4%; C. piscinae, 28.7%; C.haemolyticum 41.7%; C. pseudoviolaceum, 27.5%). The mean 16:1 w7c/16:1w6c content for all 15 isolates with 16S rRNA gene sequences identicalto either SEQ ID NO: 1 and SEQ ID NO: 2 (and/or SEQ ID NO: 3) is 45.8%with a range of 43.6% to 47.1%, which does not overlap values reportedfor any other Chromobacterium species.

EXAMPLE 3 Insecticidal Activity

C. subtsugae PRAA4-1^(T) displays broad insecticidal activity whenassayed against the lepidopteran diamondback moth or gypsy moth, and thedipteran seedcorn maggot. Meanwhile, C. vaccinii MWU205^(T) appearsactive only against the seedcorn maggot (which differs from the activityof C. vaccinii MWU205^(T) presented in WO 2015/020848 (Martin andSoby)). In contrast, C. sphagni sp. nov. strains 14B-4 and 37-2 displayinsecticidal activity comparable to C. subtsugae PRAA4-1^(T) againstdiamondback moth (see Tables 2 and 3, infra) and gypsy moth (see Table 4for strain 14B-4 only, infra), but not against seedcorn maggot (data notpresented). C. sphagni sp. nov. 37-2 also substantially inhibits growthof the lepidopteran pests tobacco hornworn and cabbage looper (seeTables 5 and 6, infra).

For diamondback moth, gypsy moth and seedcorn maggot, freeze driedpellets of insect diet are rehydrated with whole liquid cultures ofChromobacterium species (C. sphagni sp. nov. strain 14B-4 (NRRLB-67382), C. sphagni sp. nov. strain 37-2 (NRRL B-67131), C. subtsugaePRAA4-1^(T), or water (negative control)) and are fed to the insectlarvae. For gypsy moth and diamondback moth assays, the diet is asdescribed by Bell, et al. (Development of mass rearing technology. InThe gypsy moth: Research toward integrated pest management. Doane andMcManus (eds.), U.S. Department of Agriculture Technical Bulletin 1584,Washington, pp 599-633.1981). For seedcorn maggot assays an artificialmedium containing 50 g ground lima beans (Bio-Serv), 1.35 g meat andbone meal, and 10 g agar in 500 ml water is used. Hot diet is pouredinto 96-well ELISA plates with a volume of 300 μl per well, allowed tocool, and frozen. Diet is then freeze dried, and the resulting pelletsremoved from the ELISA plates. Liquid cultures are grown at 24° C. for96 hours while shaking at 200 rpm. Assays comparing toxicities ofdifferent Chromobacterium strains and species are conductedcontemporaneously. Assays for gypsy moth and diamondback larvae areconducted with 24 insects per treatment, and replicated 5 times. Thetobacco hornworm assay is run a single time with 32 larvae pertreatment; the cabbage looper assays are replicated 4 times with 24larvae per treatment. See Tables 2 and 3, below, for comparison ofinsecticidal activities of C. sphagni strains 14B-4 and 37-2 (and otherC. sphagni isolates) with C. subtsugae PRAA4-1^(T) against diamondbackmoth larvae, and see Table 4, below, for the insecticidal activities ofC. sphagni strain 14B-4 and other C. sphagni isolates against gypsy mothlarvae. The effect of consuming freeze dried diet rehydrated with wholecultures of C. sphagni sp. nov. 37-2, or C. subtsugae PRAA4-1 on growthof the lepidopteran pests, tobacco hornworm and cabbage looper, is alsoexamined. Neonate tobacco hornworm and early second instar cabbagelooper larvae are weighed after feeding on treatments for 6 days andcompared with control larvae fed freeze dried diet rehydrated with wateronly, presented below in Tables 5 and 6, respectively.

TABLE 2 Diamondback moth larvae Strain Percent pupation ± SE NegativeControl 68.6 ± 3.29 C. subtsugae PRAA4-1 28.3 ± 6.64 C. sphagni 14B-520.8 ± 6.04 C. sphagni 14B-6 15.8 ± 4.25 C. sphagni 14B-4  9.2 ± 3.33

TABLE 3 Diamondback moth larvae Strain Percent pupation ± SE NegativeControl 72.5 ± 8.8 C. sphagni 37-2 45.0 ± 9.72 C. sphagni 37-5 43.3 ±12.19 C. subtsugae PRAA4-1  9.2 ± 5.17

TABLE 4 Gypsy moth larvae Strain Percent mortality ± SE C. sphagni 14B-530.5 ± 10.43 C. subtsugae PRAA4-1 29.2 ± 9.03 C. sphagni 14B-6 28.3 ±7.84 C. sphagni 14B-4 29.2 ± 10.46 Negative Control  1.7 ± 1.7

TABLE 5 Tobacco hornworm larvae Strain Weight ± SE Negative Control156.9 ± 4.8 C. subtsugae PRAA4-1  30.3 ± 2.96 C. sphagni 37-2  23.0 ±2.15

TABLE 6 Cabbage looper larvae Strain Weight ± SE Negative Control 95.3 ±3.08 C. subtsugae PRAA4-1 62.5 ± 2.20 C. sphagni 37-2 46.4 ± 1.87

In another embodiment of this invention, a formulation of a biocontrolagent composition containing C. sphagni is a wettable powder produced bycombining a spray dried liquid fermentation media of the bacterium(containing the bacterium) with water containing emulsifiers andsurfactants that promote dispersal and suspension of the spray driedpowder in the water, and also promote wetting of leaf surfaces. Oneapplies this biocontrol agent to leaf surfaces. When an insect feeds ona treated leaf, the insect ingests the C. sphagni strain(s) and dies.

The foregoing detailed description and certain representativeembodiments and details of the invention have been presented forpurposes of illustration and description of the invention. It is notintended to be exhaustive or to limit the invention to the precise formsdisclosed. It will be apparent to practitioners skilled in the art thatmodifications and variations may be made therein without departing fromthe scope of the invention. All references cited herein are incorporatedby reference.

We, the inventors, claim as follows:
 1. A biocontrol agent useful forkilling insect larvae comprising an insecticidal composition and acarrier, wherein said insecticidal composition comprises Chromobacteriumsphagni, or media in which said C. sphagni grew, or a combinationthereof.
 2. The biocontrol agent of claim 1, wherein said C. sphagni isselected from the group consisting of C. sphagni strain 14B-4 (NRRLB-67382), C. sphagni strain 37-2 (NRRL B-67131), C. sphagni strain14B-5, C. sphagni strain 14B-6, C. sphagni strain 36-1, C. sphagnistrain 36-2, C. sphagni strain 36-3, C. sphagni strain 36-4, C. sphagnistrain 36-5, C. sphagni strain 36-6, C. sphagni strain 37-1, C. sphagnistrain 37-3, C. sphagni strain 37-4, C. sphagni strain 37-5, C. sphagnistrain 37-6, and a combination thereof.
 3. The biocontrol agent of claim1 wherein said carrier is a food of said insect larvae.
 4. A method forkilling insect larvae comprising applying said biocontrol agent of claim1 in an amount effective to kill said insect larvae, wherein saidbiocontrol agent is applied to an area in which said insect larvae arepresent or onto an object in said area.
 5. The method of claim 4,wherein said C. sphagni is selected from the group consisting of C.sphagni strain 14B-4 (NRRL B-67382), C. sphagni strain 37-2 (NRRLB-67131), C. sphagni strain 14B-5, C. sphagni strain 14B-6, C. sphagnistrain 36-1, C. sphagni strain 36-2, C. sphagni strain 36-3, C. sphagnistrain 36-4, C. sphagni strain 36-5, C. sphagni strain 36-6, C. sphagnistrain 37-1, C. sphagni strain 37-3, C. sphagni strain 37-4, C. sphagnistrain 37-5, C. sphagni strain 37-6, and a combination thereof.
 6. Themethod of claim 4, wherein said object is a plant on which said insectlarvae live or eat.
 7. The method of claim 4, wherein said insect larvaeare lepidopteran insect larvae.
 8. The method of claim 4, wherein saidcarrier is selected from the group consisting of food of said insectlarvae, water, one or more surfactants, one or more emulsifiers, one ormore alcohols, one or more oils, one or more glycerols, one or morebiological buffers, one or more ethers, one or more glycols, one or moreketones, one or more esters, one or more clays, one or more silicas, oneor more cellulosics, one or more rubber, one or more synthetic polymers,and a combination thereof.
 9. The method of claim 4, wherein saidbiocontrol agent further comprises at least one of insect larvaeattractants, adjuvants, pheromones, adhesives, and dispersants.
 10. Amethod of reducing the population of insect larvae comprising applying abiocontrol agent in an amount effective to kill said insect larvae thatingest said biocontrol agent thereby reducing the population of saidinsect larvae, wherein said biocontrol agent comprises a carrier and aninsecticidal composition comprising Chromobacterium sphagni, or media inwhich said C. sphagni grew, or a combination thereof; wherein saidbiocontrol agent is applied to an area in which said insect larvae arepresent or onto an object in said area.
 11. The method of claim 10,wherein said C. sphagni is selected from C. sphagni strain 14B-4 (NRRLB-67382), C. sphagni strain 37-2 (NRRL B-67131), C. sphagni strain14B-5, C. sphagni strain 14B-6, C. sphagni strain 36-1, C. sphagnistrain 36-2, C. sphagni strain 36-3, C. sphagni strain 36-4, C. sphagnistrain 36-5, C. sphagni strain 36-6, C. sphagni strain 37-1, C. sphagnistrain 37-3, C. sphagni strain 37-4, C. sphagni strain 37-5, C. sphagnistrain 37-6, and a combination thereof.
 12. The method of claim 10,wherein said object is a plant on which said insect larvae live or eat.13. The method of claim 10, wherein said insect larvae are lepidopteraninsect larvae.
 14. The method of claim 10, wherein said carrier isselected from the group consisting of food of said insect larvae, water,one or more surfactants, one or more emulsifiers, one or more alcohols,one or more oils, one or more glycerols, one or more biological buffers,one or more ethers, one or more glycols, one or more ketones, one ormore esters, one or more clays, one or more silicas, one or morecellulosics, one or more rubber, one or more synthetic polymers, and acombination thereof.
 15. The method of claim 10, wherein said biocontrolagent further comprises at least one of insect larvae attractants,adjuvants, pheromones, adhesives, and dispersants.
 16. A compositioncomprising at least one strain of Chromobacterium sphagni and media inwhich said C. sphagni grew.
 17. The composition of claim 16, whereinsaid C. sphagni are present in amount between approximately 10³ CFU toapproximately 10¹⁰ CFU.
 18. The composition of claim 16, wherein said C.sphagni is selected from the group consisting of C. sphagni strain 14B-4(NRRL B-67382), C. sphagni strain 37-2 (NRRL B-67131), C. sphagni strain14B-5, C. sphagni strain 14B-6, C. sphagni strain 36-1, C. sphagnistrain 36-2, C. sphagni strain 36-3, C. sphagni strain 36-4, C. sphagnistrain 36-5, C. sphagni strain 36-6, C. sphagni strain 37-1, C. sphagnistrain 37-3, C. sphagni strain 37-4, C. sphagni strain 37-5, C. sphagnistrain 37-6, and a combination thereof.
 19. The composition of claim 16,wherein said C. sphagni has a 16S rRNA gene sequence of SEQ ID NO: 1,SEQ ID NO: 2, or SEQ ID NO:
 3. 20. The composition of claim 16, whereinthe combined 16:1 w7c/16:1 w6c fatty acids in said C. sphagni rangesfrom approximately 43% to approximately 47%.